Research ArticleCell Biology

Cellular Inhibitors of Apoptosis Are Global Regulators of NF-κB and MAPK Activation by Members of the TNF Family of Receptors

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Science Signaling  20 Mar 2012:
Vol. 5, Issue 216, pp. ra22
DOI: 10.1126/scisignal.2001878
  • Fig. 1

    c-IAP proteins are required for canonical NF-κB, JNK, and p38 signaling by members of the TNFR family. (A to E) The indicated cell lines were pretreated with dimethyl sulfoxide (DMSO) as a control or with BV6 (2.5 μM) for 15 hours, after which they were treated with (A) TNF-α (20 ng/ml), (B) TL1A (100 ng/ml), (C) TWEAK (100 ng/ml), (D) LIGHT (100 ng/ml), or (E) CD40L (400 ng/ml) for the indicated times. The abundances of c-IAP1, c-IAP2, IκB, pJNK, and pp38 proteins were analyzed by Western blotting with the indicated antibodies. Actin was used as a loading control. Data are representative of at least five experiments.

  • Fig. 2

    Loss of c-IAP proteins results in reduced expression of genes regulated by members of the TNF family. (A to F) Quantitative real-time PCR analysis of the abundances of the indicated mRNAs was performed with RNA samples derived from the indicated cell lines that had been left untreated or were treated overnight with BV6 (4 μM) before being incubated with (A) TNF-α (20 ng/ml) for 4 hours, (B) TL1A (100 ng/ml) for 4 hours, (C) TWEAK (100 ng/ml) for 7 hours, (D) LIGHT (100 ng/ml) for 4 hours, (E) antibody against CD40 (500 ng/ml) for 4 and 7 hours, or (F) CD27L (100 ng/ml) for 7 hours. The abundances of the indicated mRNAs were normalized to that of RPL19, which was an internal control. Error bars represent the SD. *P < 0.05; **P < 0.01; ns, not significant. Data are representative of at least two experiments.

  • Fig. 3

    c-IAP proteins regulate the assembly of signaling complexes of TNFR family members. (A to C) HT1080, Ku812F, HT29, and Daudi cells, as indicated, were pretreated with DMSO or BV6 (3 μM) for 15 hours before being treated with FLAG–TNF-α (1 μg/ml), TL1A (1 μg/ml), His-LIGHT (1 μg/ml), FLAG-TWEAK (1 μg/ml), or antibody against CD40 for the indicated times. Cell lysates were subjected to immunoprecipitation (IP) with the indicated antibodies, and the amounts of protein in the cellular lysates and in the ligand-associated complexes were determined by Western blotting with the indicated antibodies. Data are representative of four experiments.

  • Fig. 4

    Differential effects of the signaling of TNFR family members on the stabilities of c-IAP1, c-IAP2, TRAF2, and TRAF3 proteins. (A to E) The indicated cell lines were pretreated with BV6 (3 μM) for 12 hours or MG132 (20 μM) for 30 min, after which they were incubated with (A) TNF-α (20 ng/ml), (B) TL1A (100 ng/ml), (C) LIGHT (100 ng/ml), (D) antibody against CD40 (500 ng/ml), or (E) TWEAK (100 ng/ml). The abundances of c-IAP1, c-IAP2, TRAF2, TRAF3, IκB, and pJNK proteins were determined by Western blotting analysis with the indicated antibodies. Data are representative of three experiments.

  • Fig. 5

    Proteasome and lysosome inhibitors stabilize c-IAP and TRAF proteins in cells treated with TNFR agonists. (A to C) The indicated cell lines were pretreated with MG132 (20 μM), CA-074Me (20 μM), or a combination of both inhibitors for 30 min, after which they were incubated with (A) TWEAK (100 ng/ml), (B) LIGHT (100 ng/ml), or (C) CD40L (400 ng/ml) for the indicated times. Cells were collected and lysed in 1% Triton X-100 buffer (Tr), followed by extractions of the remaining cellular pellets in 1% SDS buffer (SDS). The abundances of c-IAP1, c-IAP2, TRAF2, TRAF3, IκB, and p100 proteins were analyzed by Western blotting. Loading controls included cadherin for (A) and (B) and HSP90 for (C). Data are representative of at least three experiments.

  • Fig. 6

    Engineered membrane targeting and dimerization reduce the amounts of soluble c-IAP1 and TRAF2 proteins. (A and B) HT1080 (A) and HEK 293T (B) cells were transiently transfected with (A) control plasmid or plasmids encoding Fpk–c-IAP1 or Fpk–c-IAP1m (RING domain mutant H588A) or (B) control plasmid or plasmids encoding Myr-Fpk–c-IAP1 or Myr-Fpk–c-IAP1m. Forty-eight hours after transfection, cells were left untreated or were treated with MG132 for 30 min, after which they were treated with (A) FK1012H2 (250 nM, FK) for 1 or 3 hours or (B) were left untreated. Cellular lysates were analyzed by Western blotting with an antibody against the FLAG tag (to detect c-IAP1 constructs) and with an antibody against actin. The asterisk denotes a nonspecific band. (C and D) HEK 293T cells were transiently transfected with (C) plasmid encoding c-IAP1 together with empty plasmid (vec + c1), plasmid encoding Fpk-TRAF2 (Fpk-T2 + c1), or plasmid encoding Fpk-TRAF2ΔR (RING domain deletion) (Fpk-T2ΔR + c1) or (D) with plasmid encoding c-IAP1 together with empty plasmid (vec + c1), plasmid encoding Myr-Fpk–TRAF2 (Myr-3xFpk-T2 + c1), or plasmid encoding Myr-Fpk–TRAF2ΔR (Myr-3xFpk-T2ΔR + c1). Forty-eight hours later, cells were treated for 1 or 3 hours with FK1012H2 (250 nM, FK). Cellular lysates were analyzed with antibody against the FLAG tag (to detect c-IAP1 constructs), antibody against the Myc tag (to detect TRAF2 constructs), and antibody against actin as a loading control. Data are representative of at least three experiments.

  • Fig. 7

    BR3 signaling triggers the translocation of TRAF3 to the SDS-soluble fraction. (A) NALM-6 cells were treated with recombinant BAFF (2 μg/ml) or agonistic antibody against BR3 (αBR3, 2 μg/ml) for the indicated times. Cells were collected and lysed in CEB (Cyt.) followed by extractions of the remaining cellular pellets in 1% SDS buffer (1% SDS). The abundances of c-IAP1, TRAF2, TRAF3, HSP90, SP1, and calreticulin (Calret.) were determined by Western blotting analysis with the appropriate antibodies. (B) Purified human tonsillar B cells were pretreated with MG132 (20 μM) and CA-074Me (20 μM) for 30 min, after which they were incubated with recombinant BAFF (2 μg/ml) or agonistic antibody against BR3 (2 μg/ml) for the indicated times. Cells were collected and lysed as described for (A), and the abundances of c-IAP1, TRAF2, TRAF3, p100/p52, HSP90, SP1, and calreticulin were determined by Western blotting analysis with the appropriate antibodies. (C) WSU-FSCCL cells were treated for the indicated times with recombinant FLAG-tagged BAFF (2 μg/ml). Cell lysates were subjected to immunoprecipitation with antibody against BAFF, and protein abundances in cellular lysates and in the BAFF- and BR3-associated complexes were determined by Western blotting with the indicated antibodies. The asterisk indicates a nonspecific cross-reacting band. (D) RPMI-8226 cells were transiently transfected with empty plasmid (vector) or with plasmid encoding the indicated TRAF3 constructs. Forty-eight hours after transfection, nuclear, cytoplasmic, and membrane cellular fractions were analyzed by Western blotting with the indicated antibodies. Data are representative of three experiments.

Additional Files

  • Supplementary Materials for:

    Cellular Inhibitors of Apoptosis Are Global Regulators of NF-κB and MAPK Activation by Members of the TNF Family of Receptors

    Eugene Varfolomeev, Tatiana Goncharov, Heather Maecker, Kerry Zobel, László G. Kömüves, Kurt Deshayes, Domagoj Vucic*

    *To whom correspondence should be addressed. E-mail: domagoj{at}gene.com

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Characterization of commercial antibodies against c-IAP1, c-IAP2, TRAF2, TRAF3, TRAF6, IKK2, NEMO, TAK1, and HOIP.
    • Fig. S2. Characterization of the cell surface expression of members of the TNFR family.
    • Fig. S3. c-IAPs are required for canonical NF-κB, JNK, and p38 activation by TNF-α, TWEAK, LIGHT, CD27L, and CD30L.
    • Fig. S4. TRAF6 and c-IAP proteins mediate CD40-dependent activation of the canonical NF-κB pathway.
    • Fig. S5. TNF-α–, TWEAK-, and LIGHT-stimulated NF-κB and JNK activation depends on c-IAP1 and c-IAP2.
    • Fig. S6. Loss of c-IAP proteins results in reduced expression of genes regulated by members of the TNF family.
    • Fig. S7. c-IAP proteins regulate the function of complexes involving TNFR family members.
    • Fig. S8. TNF-α–, TWEAK-, and LIGHT-stimulated NF-κB and JNK activation depends on NEMO and HOIP.
    • Fig. S9. Differential effects of the signaling of TNFR family members on the stability of c-IAP1, c-IAP2, TRAF2, and TRAF3 proteins.
    • Fig. S10. Proteasome and lysosome inhibitors stabilize c-IAP and TRAF proteins during signaling by TNF family members.
    • Fig. S11. Membrane targeting of TRAF2 or c-IAP1 reduces their cytoplasmic abundance and enables noncanonical NF-κB signaling.
    • Fig. S12. BR3 signaling triggers the translocation of TRAF3 to the insoluble fraction.

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    Citation: E. Varfolomeev, T. Goncharov, H. Maecker, K. Zobel, L. G. Kömüves, K. Deshayes, D. Vucic, Cellular Inhibitors of Apoptosis Are Global Regulators of NF-κB and MAPK Activation by Members of the TNF Family of Receptors. Sci. Signal. 5, ra22 (2012).

    © 2012 American Association for the Advancement of Science

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